Infection of target cells by HIV-1 is initially dependent on the interaction of its envelope glycoproteins with CD4 and a seven transmembrane-spanning G protein coupled receptor (GPCR). Among the several identified GPCR coreceptors, the chemokine receptors CCR5 and CXCR4 are by far the most commonly used by HIV-1. As a consequence, these receptors have been identified as important new targets for anti-HIV treatment (1). Infection by HIV strains is inhibited by the natural ligands of the coreceptors they use. Among these are MIP-1α (CCL3), MIP-1β (CCL4) and RANTES (CCL5) for CCR5 (2), CCR1 and CCR3, and SDF-1 (CXCL12) for CXCR4 (3). These ligands are chemokines, a group of small (8–12 kDa), structurally similar proteins with important roles in development and inflammation (4).
Chemokines have the ability to recruit and activate a wide variety of proinflammatory cell types, and RANTES has been shown to elicit an inflammatory response in vivo. RANTES, along with the natural ligands for the CCR5, CCR1 and CCR3 chemokine receptor, MIP-1.alpha., MIP-1.beta., were found to inhibit human immune deficiency virus type-1 (“HIV-1”) infection (2), leading to the identification of CCR5 as the major co-receptor for primary isoiates of HIV-1, HIV-2 and SIV-1 (11). However, although RANTES consistently inhibits HIV-1 replication in periphera! blood mononuclear cells, it does not block infection of primary macrophage cultures, which suggests that RANTES would not influence HIV replication in non-lymphocyte cell types.
Structurally, chemokines consist of a tightly folded core from which extends a flexible region comprising approximately ten amino terminal residues. The last few C-terminal residues themselves are mobile and exposed to the solution (5). The description of a C-terminally anchored chemokine (6), as well as C-terminally conjugated chemokines (7), suggests that relatively large entities can be accommodated at the C-terminal of chemokines without compromising biological activity. According to the current model for interaction between chemokines and their receptors (8), the flexible amino terminal region is necessary for receptor activation, whereas the tigthly-folded core contains structures necessary for receptor recognition. This “two-site” model for interaction is supported by the description of chemokine variants with altered signaling activity that were obtained by modification at the amino terminus (9–11).
These above-mentioned amino-terminally modified chemokines include aminooxypentane(AOP)—RANTES, an extremely potent inhibitor of monocytotropic CCR5-dependent (R5-tropic) strains of HIV-1 (11). The strongly enhanced anti-HIV activity of AOP-RANTES is due to its ability to bring about profound and sustained downmodulation of cell surface CCR5 (12) in a process that probably involves the induction of receptor endocytosis (13). It has recently been shown that several other amino terminally modified chemokine analogues with enhanced anti-HIV activity appear to act via similar mechanisms (14, 15).
More generally, the pivotal role of chemokines in inflammatory disease is now well established (45), and the chemokine/chemokine receptor network has been validated as a target for therapeutic intervention (46). Agents that either antagonize the activity of appropriate chemokines or achieve blockade of their receptors could be used to treat inflammatory diseases. Specific examples of clinical conditions in which either RANTES or CCR5, CCR1 and/or CCR3 have been identified as key pathogenic factors (and hence where antagonists or inhibitors would be of clinical use) include asthma, organ transplant rejection, immune complex glomerulonephritis multiple sclerosis, rheumatoid arthritis, allergic rhinitis, atopic dermatitis, viral diseases and atheroma/atheroschleosis.
In addition, a link between the chemokine/chemokine receptor network and cancer has recently been established (47) indicating roles for both RANTES (48) and CCR5 (49) in the development and maintenance of tumors. Hence agents that either antagonize the activity of appropriate chemokines or achieve blockade of their receptors could be used to treat cancer.
With this in mind, the inventors reasoned that the amino terminus of RANTES would be a promising region in which to introduce diversity as part of a search for RANTES mutants with further improved activity.
Phage display is a known technology which presents a means by which diversity in a protein structure can be coupled to rapid selection of a desired phenotype. Conventional phage display involves the panning of a library of encoded and displayed ligands against a purified target which is attached to the solid phase, usually with the displayed ligands expressed as fusion proteins, N-terminal to the gene-three protein of the phage (16). This technique has been widely applied for over a decade, allowing the isolation of ligands with high affinity and specificity for many different targets (17).
Conventional phage display techniques are not easily applied for the selection of ligands of integral membrane proteins like GPCRs. As a consequence, new phage display strategies have been adopted to allow the selection of ligands that bind to targets presented on the cell surface (18). In addition, if living cells are used for the presentation of selection targets it is possible to select for phage particles internalized by receptor-mediated endocytosis (19) and thus selecting those acting via a downmodulation of the receptor. In addition, the use of living cells allows the selection of phages not internalized by receptor-mediated endocytosis but having a selective affinity for GPCRs and acting as agonists or antagonists thereof.
A novel approach for the directed evolution of chemokines, based on the use of phage display together with living cells, would therefore appear as a powerful improvement.